Progress has been made in several of the aims of the project, in particular on the role of TLR signaling in regulating proliferation and survival of fibroblasts and dendritic cells, on the regulation of cytokine production from human DC, on the role of plasmacytoid DC in regulating oral tolerance, on the effect of NK cells on Th1 differentiation in Toxoplasma Gondii infection, role of TNF, MyD88 and IL-18 in colitis-dependent carcinogenesis. Cell proliferation and survival induced by TLRs is antagonized by type I Interferons (PNAS 2007 19:8047-52). TRIF is an adaptor protein associated with the signalling by TLR3 and TLR4 for the induction of type-I IFNs. We demonstrated a novel mechanism by which TLR signalling controls cell proliferation and survival. We showed that TLR3 and TLR4 can induce cell cycle entry via TRIF, which targets the cell cycle inhibitor p27kip1 for re-localisation, phosphorylation by cyclin/cdk complexes, and proteasome degradation. These events are antagonized by type-I IFN induced by the TRIF pathway. Furthermore, in human dendritic cells treated with TLR3, TLR4 or TLR5 ligands, we demonstrated that interferon signalling modulates p27kip1 degradation and apoptosis, identifying a novel immunoregulatory switching function of type-I IFNs. These findings revealed a new function of TLR signalling in cell proliferation and survival. These findings are now being extended to analyze the role of IFN-mediated inhibition of AKT activation and signaling in proinflammatory cytokine production by DC. Differential regulation of interleukin-12 and interleukin-23 production in human dendritic cells and role of the Il-12 family of cyutokines in carcinogenesis and cancer therapy. (J Exp Med. 2008, 205:1447-61). We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand beta-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) gamma strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by beta-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor beta, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4(+) T cells, IL-1beta, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection. We are now continuing these studies by investigating the mechanisms by which the beta-glucan receptor induce IL_1 production through Caspase 1 induction and the dependence of IL-23 and IL-12 production on endogeneous IL-1. We are also investigating the molecular control of IL-12, IL-23, IL-27, and IL-35 and the role of these cytokines in tumor control and carcinogenesis. Hepatic plasmacytoid dendritic cells contribute to orally induced CD8+ T cell tolerance (Immunity. 2008; 9:464-75). The liver is thought to contribute to systemic T cell tolerance to orally absorbed antigens, although the precise mechanism of its tolerogenic effect is unclear. Here we show that the liver is a site of oral antigen presentation and that hepatic dendritic dells (DC) can tolerize mice to subsequent CD8+ T cell priming, in a model of hapten-specific contact sensitivity (CHS). The tolerogenic potential of liver DC is confined to plasmacytoid DC (pDC), is enhanced by exposure to hapten, and requires CD4+ T cells. Finally, in vivo depletion of pDC abrogated oral tolerance and restored hapten-specific CD8+ T cell and CHS responses. Thus, pDC are tolerogenic and play an essential role in oral tolerance. These studies are now being extended to analyze the role of liver pDC in other model of tolerance and in particular in testing whether pDC are require to induce TNBS tolerance in a model of colitis and carcinogenesis. Toxoplasma gondii infected TAP1 deficient mice display impaired NK cell IFN-gamma production leading to defective CD4+ T cell priming and increased mortality (J Exp Med. 2007, 204:2591-602). To investigate if Transporter Associated with Antigen Processing (TAP)1 is required for CD8+ T cell mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP1-/-, CD8-/- and wild-type (WT) mice to infection with the parasite. Surprisingly TAP1-/- mice displayed greater susceptibility than either CD8-/- or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP1-/- mice correlated with a reduction in the frequency of activated and IFN-gamma-producing CD4+ T cells. Interestingly, infected TAP1-/- mice showed a reduced frequency of IFN-gamma producing natural killer (NK) cells relative to that of WT controls, and after NK cell-depletion both CD8-/- and WT mice succumbed to infection with the same kinetics as TAP1-/- animals and displayed impaired CD4+T cell IFN-gamma responses. Together, these results reveal a previously unappreciated role for TAP1 in the induction of IFN-gamma &#61472;producing NK cells and provide the first demonstration of the function of this cell population in the priming of CD4+ T lymphocyte responses to T. gondii infection. These studies are now being extended to understand the difference between the TAP1-/- and beta2microglobulin-/- in this function of NK cells and the possible role of NKT cells in the regulation of NK activity. Innate resistance and pro-inflammatory cytokines in carcinogenesis. A very extensive investigation has been initiated to study the role of inflammatory receptors and cytokines in skin and colon chemical carcinogenesis. Following our early observation that MyD88-/- are resistant to skin carcinogenesis, we have now showed that MyD88 expression is required both in radioresistant skin cells as well as in hematopoieitc cells and that MyD88-/- keratinocytes are very altered in their gene expression pattern after Ras transformation in vitro, suggesting that this altered pattern of gene expression may be responsible for the lack of polyps formation in response to chemical carcinogenesis. In the colon, MyD88 deficiency resulted in an increased susceptibility to DSS induced colitis that surprisingly was associated with a highly increased susceptibility to chemically induced carcinogenesis. The MyD88 phenotype was in part due to lack of signaling through the IL-18 receptor in the MyD88-/- cells. We also observed that TNF is required for colitis-dependent carcinogenesis but, interestingly, TNF produced by the enterocytes was required for carcinogenesis whereas T cells and macrophages produced TNF h [summary truncated at 7800 characters]